2.
PCR is an abbreviation for "polymerase chain
reaction". PCR is a method for amplifying DNA. So if
you have one molecule of DNA you can increase it to
thousands of molecules within a few hours. This is
achieved using an enzyme called DNA polymerase
and the raw material or "building blocks" of DNA.
POLYMERASE CAHIN REACTION
5.
Roles of PCR
• Forensic applications
• Infectious disease
• Medical applications
• Research applications
• Disease diagnosis
• Detecting infectious agents
• cancer diagnostics
6.
Polymerase chain reaction (PCR)
optimization
• In practice, PCR can fail for various
reasons, in part due to its sensitivity to
contamination causing amplification of
spurious DNA products. Because of this,
a number of techniques and procedures
have been developed for optimizing PCR
conditions.
7.
Contamination and PCR
• The PCR method is extremely sensitive, requiring
only a few DNA molecules in a single reaction for
amplification across several orders of magnitude.
• One lab area is dedicated to preparation and
handling of pre-PCR reagents and the setup of the
PCR reaction, and another area to post-PCR
processing, such as gel electrophoresis or PCR
product purification.
8. Hairpins
Secondary structures in the DNA can
result in folding or knotting of DNA
template or primers, leading to decreased
product yield or failure of the reaction
addition of DMSO or glycerol to the PCR
to minimize secondary structures in the
DNA template are used in the
optimization of PCRs
9.
Magnesium is required as a co-
factor for thermostable DNA
polymeraseTaq polymerase is a
magnesium-dependent enzyme
and determining the optimum
concentration to use is critical to
the success of the PCR reaction
Magnesium concentration
10.
Size and other limitations
PCR works readily with a
DNA template of up to two to
three thousand base pairs in
length. However, above this
size, product yields often
decrease, as with increasing
length
11.
Other Measures
• Use of disposable plastic ware
• Thoroughly cleaning the work surface between
reaction setups
• Using pipettes with filter tips
• Wearing fresh laboratory gloves
